The FDA approval of the proteasome inhibitor bortezomib/VELCADE(r) for the treatment of multiple myeloma and other hematological malignancies demonstrates that the ubiquitin pathway is a promising source of new drugs for the treatment of cancer and perhaps other diseases. Bortezomib, a proteasome inhibitor, exerts a global effect and its use is thus limited by a serious toxicity profile. Efforts are underway to ind ubiquitin pathway enzyme targets acting more selectively than the proteasome with the aim of developing drugs with improved toxicity profiles and therapeutic indices. Among these enzymes are the ubiquitin E1 activating enzyme, E2 conjugating enzyme, E3 ligase and Deubiquitylases (DUBs). DUBs act on limited numbers of target proteins and are linked biochemically and genetically to various diseases, including cancer. These enzymes are proteases and are considered druggable, and various pharmaceutical and biotech companies, including Progenra have identified novel, selective DUB inhibitors. Despite this progress, no DUB inhibitors have progressed to clinical evaluation. Part of the reason for this situation may be that improved screening assays are needed to identify hits for preclinical development that inhibit the DUB acting upon its in situ substrate. The project described in this application is the development of physiologically relevant substrate- based assay that introduces a significant improvement over all of the other assays currently in use to screen for DUB inhibitors. The assay is novel, homogeneous, HTS-compliant, and utilizes specific polyubiquitylated DUB substrates. The therapeutically relevant DUB/ubiquitylated substrate pairs USP7/HDM2/p53 (involved in cancer cell proliferation) will be prepared and purified, the assay configured, and a pilot screen for inhibitors will be conducted. This novel assay platform, when validated, will be expanded to include additional DUBs. The ultimate commercial goal of the proposed work is a robust, homogenous assay format that is suitable for HTS and customizable to various DUB/substrate pairs, allowing rapid progress in drug discovery for known and emerging DUB-mediated diseases.